This invention relates to polypeptide hormone analogues that exhibits enhanced pharmaceutical properties, such as increased increased thermodynamic stability, augmented resistance to thermal fibrillation above room temperature, and decreased mitogenicity. More particularly, this invention relates to insulin analogues that are modified by substitution of non-standard Phenylalanine at position 24 of the B-chain (designated herein position B24). Such non-standard sequences may optionally contain standard amino-acid substitutions at other sites in the A or B-chains of an insulin analogue. Even more particularly, this invention relates to insulin analogues that are characterized by the incorporation of the element fluorine into phynylalanine at position B24 of the insulin analogue. Fluorine is distinguished from the ordinary constituents of proteins by its atomic radius, electronegativity, stereoelectronic distribution of partial charges, transmitted effects on the stereoelectronic properties of neighboring atoms in a molecule.
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. Naturally occurring proteins—as encoded in the genomes of human beings, other mammals, vertebrate organisms, invertebrate organisms, or eukaryotic cells in general—often confer multiple biological activities. A benefit of non-standard proteins would be to achieve selective activity, such as action in one organ, tissue, or cell type and not in another. Yet another benefit would be decreased risk of an unintended and unfavorable side effect, such as promotion of the growth of cancer cells. An example of a therapeutic protein is provided by insulin. Wild-type human insulin and insulin molecules encoded in the genomes of other mammals bind to insulin receptors is multiple organs and diverse types of cells, irrespective of the receptor isoform generated by alternative modes of RNA splicing or by alternative patterns of post-translational glycosylation. Such ubiquitious binding leads to opposing biological signals, such as an anabolic effect of insulin signaling in the liver, muscle, and fat but a catabolic effect of insulin signaling in the mammalian brain as mediated by specific insulin-stimulated and insulin-modulated circuits in the hypothalamus.
An example of a further medical benefit would be optimization of the stability of a protein toward unfolding or degradation. Such a societal benefit would be enhanced by the engineering of proteins more refractory than standard proteins with respect to degradation at or above room temperature for use in regions of the developing world where electricity and refrigeration are not consistently available. Analogues of insulin containing non-standard amino-acid substitutions may in principle exhibit superior properties with respect to resistance to thermal degradation or mitogenicity. The challenge posed by its physical degradation is deepened by the pending epidemic of diabetes mellitus in Africa and Asia. Because fibrillation poses the major route of degradation above room temperature, the design of fibrillation-resistant formulations may enhance the safety and efficacy of insulin replacement therapy in such challenged regions.
The utility of some halogen substitutions in amino acids is well established in medicinal chemistry. Indeed, fluorinated functional groups are critical to the efficacy of such widely prescribed small molecules as atorvastatin (Liptor™), an inhibitor of cholesterol biosynthesis, and fluoxetine hydrochloride (Prozac™), a selective serotonin reuptake inhibitor used in the treatment of depression and other affective disorders. Although the atomic radius of fluorine is similar to that of hydrogen, its large inductive effects modify the stereo-electronic properties of these drugs, in turn enhancing their biological activities. Such observations have motivated the study of fluorinated amino acids in proteins. Similar considerations of physical organic chemistry pertain to the incorporation of larger halogen atoms, such as chlorine and bromine. The small molecule montelukast sodium (Singulair™) is a leukotriene inhibitor whose pharmaceutical properties are enhanced by covalent incorporation of a chlorine atom.
Attention has previously focused on the use of multiply fluorinated aliphatic side chains (such as trifluoro-γ-CF3-Val, trifluoro-δ-CF3-Val, trifluoro-δ-CF3-Ile, hexafluoro-γ1,2-CF3-Val, and hexafluoro-δ1,2-CF3-Leu) to maximize the gain in hydrophobicity associated with this modification. An example is provided by the stabilization of a model α-helical motif, the homodimeric coiled coil. Its interfacial aliphatic chains were simultaneously substituted by trifluoro-analogs, creating a fluorous core whose stability is enhanced by 0.3-2.0 kcal/mole. The degree of stabilization per fluorine atom is <0.1 kcal/mole. More marked stabilization per fluorine atom has been achieved in an unrelated α-helical domain by substitution of a single internal Phe by pentafluoro-Phe (F5-Phe)1 (ΔΔGu 0.6 kcal/mole per five fluorine atoms). Stabilization occurs only at one specific position in the protein, suggesting that its mechanism requires a particular spatial environment. The structure of the F5-Phe-modified domain is identical to that of the unmodified domain. Structural stabilization however, is only a portion of the requirements for a biologically active polypeptide. At least a significant portion of the activity must also be maintained.
An extensive literature describes the use of fluorine labels in proteins as 19F-NMR probe. Whereas such labels are widely regarded as non-perturbing, applications of fluorinated amino acids in protein engineering seek to exploit their altered physicochemical properties. Studies of a model α-helical fold (the villin headpiece subdomain) containing single F5-Phe substitutions have demonstrated that whether and to what extent such a modification may affect protein stability depends in detail on structural environment. Indeed, the stability of this model fold was enhanced by an F5-Phe substitution only at one of the seven sites tested in the core. It should be noted however, that this stabilizing effect was only demonstrated in a nonstandard polypeptide analogue containing a sulfur atom in the main chain near the site of fluorination; in our hands the self-same F5-Phe substitution in villin headpiece subdomain with native polypeptide main chain has no effect on its stability. Thus, to our knowledge, bona fide data demonstrating enhancement of protein stability due to halogenation of an aromatic residue in an otherwise native protein has not previously been reported. These observations suggest that a generally hydrophobic environment does not in itself assure that the modification will be stabilizing. Because protein interiors are often stabilized by aromatic-aromatic interactions with a specific distance- and angular dependence, a subset of stabilizing F5-Phe substitutions may adopt particularly favorable perfluoroaryl/aryl geometries. Such interactions arise from the asymmetric distribution of partial charges in these aromatic systems. Modulation of the chemical, physical, and biological properties of proteins by the site-specific incorporation of chlorine or bromine atoms into modified amino acids are less well characterized in the scientific literature than are the above effects of incorporation of fluorine atoms.
Aromatic side chains may engage in a variety of weakly polar interactions, involving not only neighboring aromatic rings but also other sources of positive- or negative electrostatic potential. Examples include main-chain carbonyl- and amide groups in peptide bonds.
Administration of insulin has long been established as a treatment for diabetes mellitus. Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues and a B-chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B-chain (residue B30) to the N-terminal residue of the A chain (FIG. 1A). Although the structure of proinsulin has not been determined, a variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide (FIG. 1B). Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19; FIGS. 1A and 1B) is thought to be coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin assembles to form soluble Zn2+-coordinated hexamers shortly after export from ER to the Golgi apparatus. Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). The sequence of insulin is shown in schematic form in FIG. 1C. Individual residues are indicated by the identity of the amino acid (typically using a standard three-letter code), the chain and sequence position (typically as a superscript). Pertinent to the present invention is the designation of individual hydrogen atoms attached to carbon atoms in the aromatic ring of Phenylalanine, designated ring positions 2-6.
Insulin functions in the bloodstream as a monomer, and yet it is the monomer that is believed to be most susceptible to fibrillation and most forms of chemical degradation. The structure of an insulin monomer, characterized in solution by NMR, is shown in FIG. 1D. The A-chain consists of an N-terminal α-helix (residues A1-A8), non-canonical turn (A9-A12), second α-helix (A12-A18), and C-terminal extension (A19-A21). The B-chain contains an N-terminal arm (B1-B6), β-turn (B7-B10), central α-helix (B9-B19), β-turn (B20-B23), β-strand (B24-B28), and flexible C-terminal residues B29-B30. The two chains pack to form a compact globular domain stabilized by three disulfide bridges (cystines A6-A11, A7-B7, and A20-B19).
Absorption of regular insulin is limited by the kinetic lifetime of the Zn-insulin hexamer, whose disassembly to smaller dimers and monomers is required to enable transit through the endothelial lining of capillaries. The essential idea underlying the design of Humalog® and Novolog® is to accelerate disassembly. This is accomplished by destabilization of the classical dimer-forming surface (the C-terminal anti-parallel β-sheet). Humalog® contains substitutions ProB28→Lys and LysB29→Pro, an inversion that mimics the sequence of IGF-I. Novolog® contains the substitution ProB28→Asp. Although the substitutions impair dimerization, the analogs are competent for assembly of a phenol- or meta-cresol-stabilized zinc hexamer. This assembly protects the analog from fibrillation in the vial, but following subcutaneous injection, the hexamer rapidly dissociates as the phenol (or m-cresol) and zinc ions diffuse away. The instability of these analogs underlies their reduced shelf life on dilution by the patient or health-care provider. It would be useful for an insulin analogue to augment the intrinsic stability of the insulin monomer while retaining the variant dimer-related β-sheet of Humalog®.
Use of zinc insulin hexamers during storage is known and represents a classical strategy to retard physical degradation and chemical degradation of a formulation in the vial or in the reservoir of a pump. Because the zinc insulin hexamer is too large for immediate passage into capillaries, the rate of absorption of insulin after subcutaneous injection is limited by the time required for dissociation of hexamers into smaller dimers and monomer units. Therefore, it would advantageous for an insulin analogue to be both (a) competent to permit hexamer assembly at high protein concentration (as in a vial or pump) and yet (b) sufficiently destabilized at the dimer interface to exhibit accelerated disassembly—hence predicting ultra-rapid absorption from the subcutaneous depot. These structural goals walk a fine line between stability (during storage) and instability (following injection).
Amino-acid substitutions in insulin have been investigated for effects on thermodynamic stability and biological activity. No consistent relationship has been observed between stability and activity. Whereas some substitutions that enhance thermodynamic stability also enhance binding to the insulin receptor, other substitutions that enhance stability impede such binding. The effects of substitution of ThrA8 by several other amino acids has been investigated in wild-type human insulin and in the context of an engineered insulin monomer containing three unrelated substitutions in the B-chain (HisB10→Asp, ProB28→Lys, and LysB29→Pro) have been reported. Examples are also known in the art of substitutions that accelerate or delay the time course of absorption. Such substitutions (such as AspB28 in Novalog® and [LysB28, ProB29] in Humalog®) can be and often are associated with more rapid fibrillation and poorer physical stability. Indeed, a series of ten analogs of human insulin have been tested for susceptibility to fibrillation, including AspB28-insulin and AspB10-insulin. All ten were found to be more susceptible to fibrillation at pH 7.4 and 37° C. than is human insulin. The ten substitutions were located at diverse sites in the insulin molecule and are likely to be associated with a wide variation of changes in classical thermodynamic stability. Although a range of effects has been observed, no correlation exists between activity and thermodynamic stability.
Insulin is a small globular protein that is highly amenable to chemical synthesis and semi-synthesis, which facilitates the incorporation of nonstandard side chains. Insulin contains three phenylalanine residues (positions B1, B24, and B25). Conserved among vertebrate insulins and insulin-like growth factors, the aromatic ring of PheB24 packs against (but not within) the hydrophobic core to stabilize the super-secondary structure of the B-chain. PheB24 lies at the classical receptor-binding surface and has been proposed to direct a change in conformation on receptor binding.
The present theory of protein fibrillation posits that the mechanism of fibrillation proceeds via a partially folded intermediate state, which in turn aggregates to form an amyloidogenic nucleus. In this theory, it is possible that amino-acid substitutions that stabilize the native state may or may not stabilize the partially folded intermediate state and may or may not increase (or decrease) the free-energy barrier between the native state and the intermediate state. Therefore, the current theory indicates that the tendency of a given amino-acid substitution in the insulin molecule to increase or decrease the risk of fibrillation is highly unpredictable.
Fibrillation, which is a serious concern in the manufacture, storage and use of insulin and insulin analogues for diabetes treatment, is enhanced with higher temperature, lower pH, agitation, or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, diabetic individuals optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or an insulin analogue can be a particular concern for diabetic patients utilizing an external insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient's body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable blood glucose level fluctuations or even dangerous hyperglycemia. At least one recent report has indicated that lispro insulin (an analogue in which residues B28 and B29 are interchanged relative to their positions in wild-type human insulin; trade name Humalog®) may be particularly susceptible to fibrillation and resulting obstruction of insulin pump catheters. Insulin exhibits an increase in degradation rate of 10-fold or more for each 10° C. increment in temperature above 25° C.; accordingly, guidelines call for storage at temperatures <30° C. and preferably with refrigeration. The propensity of current products Humalog®, Novalog®, and Apidra® to form fibrils at or above room temperature is exacerbated on dilution, as may be used in the treatment of Type I diabetes mellitus in children or adults with low body mass. Accordingly, the shelf life of such diluted pharmaceutical formulations in use at room temperature is reduced in accordance with instructions contained in package inserts regarding disposal of diluted insulin analogue formulations. Fibrillation of basal insulin analogues formulated as soluble solutions at pH less than 5 (such as Lantus® (Sanofi-Aventis), which contains an unbuffered solution of insulin glargine and zinc ions at pH 4.0) also can limit their self lives due to physical degradation at or above room temperature; the acidic conditions employed in such formulations impairs insulin self-assembly and weakens the binding of zinc ions, reducing the extent to which the insulin analogues can be protected by sequestration within zinc-protein assemblies.
Insulin fibrillation is an even greater concern in implantable insulin pumps, where the insulin would be contained within the implant for 1-3 months at high concentration and at physiological temperature (i.e. 37° C.), rather than at ambient temperature as with an external pump. Additionally, the agitation caused by normal movement would also tend to accelerate fibrillation of insulin. In spite of the increased potential for insulin fibrillation, implantable insulin pumps are still the subject of research efforts, due to the potential advantages of such systems. These advantages include intraperitoneal delivery of insulin to the portal circulatory system, which mimics normal physiological delivery of insulin more closely than subcutaneous injection, which provides insulin to the patient via the systemic circulatory system. Intraperitoneal delivery provides more rapid and consistent absorption of insulin compared to subcutaneous injection, which can provide variable absorption and degradation from one injection site to another. Administration of insulin via an implantable pump also potentially provides increased patient convenience. Whereas efforts to prevent fibrillation, such as by addition of a surfactant to the reservoir, have provided some improvement, these improvements have heretofore been considered insufficient to allow reliable usage of an implanted insulin pump in diabetic patients outside of strictly monitored clinical trials.
As noted above, the developing world faces a challenge regarding the safe storage, delivery, and use of drugs and vaccines. This challenge complicates the use of temperature-sensitive insulin formulations in regions of Africa and Asia lacking consistent access to electricity and refrigeration, a challenge likely to be deepened by the pending epidemic of diabetes in the developing world. Insulin exhibits an increase in degradation rate of 10-fold or more for each 10° C. increment in temperature above 25° C., and guidelines call for storage at temperatures <30° C. and preferably with refrigeration. At higher temperatures insulin undergoes both chemical degradation (changes in covalent structure such as formation of iso-aspartic acid, rearrangement of disulfide bridges, and formation of covalent polymers) and physical degradation (non-native aggregation and fibrillation. A major goal of conventional insulin replacement therapy in patients with diabetes mellitus is tight control of the blood glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions below the normal range are associated with immediate adrenergic or neuroglycopenic symptoms, which in severe episodes lead to convulsions, coma, and death. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinaphthy, blindness, and renal failure. The classical paradigm of insulin action has focused on organ-specific functions of adipocytes (where insulin regulates storage of fuels in the form of tryglyceride droplets), the liver (where insulin regulates the production of glucose via gluconeogenesis and regulates the storage of fuel in the form of glycogen) and muscle (where insulin regulates the influx of glucose from the bloodstream via trafficking to the plasma membrane of GLUT4) as the target tissues of the hormone. Recent research has revealed, however, that insulin has physiological roles in other organs and tissues, such as in the hypothalamus of the brain, wherein insulin-responsive neural circuitry influences hepatic metabolism, appetite, satiety, and possibly the set point for ideal body weight. In addition to the brain as a site of hormonal regulation of overall metabolism and feeding behaviors, resistance of the brain to the action of insulin is proposed to contribute to the decline of cognitive function in Alzheimer's Disease and may be a molecular mechanism contributing to its pathogenesis. Although the human genome contains a single gene encoding the insulin receptor, a transmembrane protein containing a cytoplasmic tyrosine-kinase domain, its pre-messanger RNA undergoes alternative splicing to yield distinct A and B isoforms, whose fractional distribution may differ from organ to organ and whose signaling functions may differ within the same cells. The A and B isoforms (designated IR-A and IR-B) differ in affinity for insulin, and only IR-A (lacking a peptide domain in the alpha subunit encoded by exon 11) binds IGF-II with high affinity. Insulin analogues have been described by investigators at Novo-Nordisk that differ from wild-type insulin in the ratio of respective affinities for IR-A and IR-B. In addition, the insulin receptor undergoes a complex pattern of N- and O-linked glycosylation, which may vary from organ to organ or from tissue to tissue; it is not know whether such potential variation can influence the relative binding affinities and signaling potencies of insulin analogues. A further source of organ- or cell-type-specific variation in response to insulin or an insulin analogue could be the co-expression of other membrane proteins or receptors that structurally or functionally interact with the insulin receptor to modulate its signaling effects; such an interacting receptor has been described in the liver due to functional interactions between IR and the MET proto-oncoprotein, the receptor for hepatic growth factor. It is also possible that kinetic and thermodynamic features of how insulin binds to the insulin receptor affect the extent and efficiency of insulin signaling differently in different organs. Together, the complex biochemistry and cell biology of the insulin receptor make it difficult to predict the physiological effects of an insulin analogue based solely on its receptor-binding affinity in vitro.
The relative contributions of organ-specific insulin pathways may differ among mammals. In rodents, for example, insulin regulates hepatic gluconeogenesis in large part by an indirect mechanism involving insulin-responsive neural circuits in the hypothalamus, which modulate vagal inputs to the liver. By contrast, in dogs the major mechanism by which insulin regulates hepatic gluconeogenesis is through the binding of insulin to insulin receptors expressed on the surface of hepatocytes. Whereas the amino-acid sequence of the rodent insulin receptor in rodents is almost identical to the amino-acid sequence of the canine insulin receptor, their physiological mode of regulation of the liver (direct or indirect) is not equivalent. In both animals insulin is nonetheless thought to function in the hypothalamus to regulate appetite and satiety in conjunction with leptin, melanocortin, and neuropeptide Y. Comparison of the regulatory pathways of insulin in these two mammals raises the possibility that a putative organic-specific insulin analogue, should such a molecule be found, could exhibit different integrated hypoglycemic potencies in different animals and different integrated hypoglycemic potencies in the same animal depending on its physiological status in the period following subcutaneous injection (i.e., depending on the extent to which hepatic glucose production and output is contributing to the maintenance or elevation of blood glucose concentration).
Non-classical functions of insulin extrinsic to adipose tissue, liver, and muscle may have beneficial or undesired effects. An example of a beneficial effect is provided by the action of insulin in the brain to suppress appetite. An example of an undesired effect of insulin is its ability to stimulate the proliferation of cancer cells. Wild-type insulin exhibits mitogenic activity, especially at the high concentrations needed to treat patients with diabetes mellitus who also exhibit marked insulin resistance. Indeed, hyperinsulinemia (whether endogenous due to beta-cell hypersecretion or as a consequence of insulin replacement therapy) is associated on an epidemiological level with an increased risk of several common tumors, including breast cancer and colon cancer. Although molecular mechanisms of such mitogenic signaling are not understood in detail, proliferation of human cancer cell lines is increased by insulin analogs that exhibit higher affinity for IR-A and the homologous Type 1 insulin-like growth factor receptor or that exhibit prolonged residence times on these receptors. It would be desirable, therefore, to invent an insulin analogue with negligible mitogenicity that nonetheless retains at least a portion of the glucose-lowering effect of wild-type insulin. More generally, there is a need for an insulin analogue that displays increased thermodynamic stability and increased resistance to fibrillation above room temperature while exhibiting at least a subset of the multiple organ-specific biological activities of the corresponding wild-type insulin.
Amino-acid substitutions have been described in insulin that stabilize the protein but augment its binding to the insulin receptor (IR) and its cross-binding to the homologous receptor for insulin-like growth factors (IGFR) in such a way as to confer a risk of carcinogenesis. An example known in the art is provided by the substitution of HisB10 by aspartic acid. Although AspB10-insulin exhibits favorable pharmaceutical properties with respect to stability and pharmacokinetics, its enhanced receptor-binding properties were associated with tumorigenesis in Sprague-Dawley rats. Although there are many potential substitutions in the A- or B-chains that can be introduced into AspB10-insulin or related analogues to reduce its binding to IR and IGFR to levels similar to that of human insulin, such substitutions generally impair the stability of insulin (or insulin analogues) and increase its susceptibility to chemical and physical degradation. It would be desirable to discover a method of modification of insulin and of insulin analogues that enabled “tuning” of receptor-binding affinities while at the same time enhancing stability and resistance to fibrillation. Such applications would require a set of stabilizing modifications that reduce binding to IR and IGFR to varying extent so as to offset the potential carcinogenicity of analogues that are super-active in their receptor-binding properties.
Therefore, there is a need for alternative insulin analogues, including those that are stabilized during storage while maintaining at least a portion of the biological activity of the analogue.